During the isolation of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) from post-mortem human brain tissue, researchers find that the peptide is rapidly degraded by peptidases in 1 minute at 37 C. Detailed analysis of the peptide cleavage pattern of Met-enkephalin is investigated with two candidate enzymes. Using the drug bestatin, the investigators found no detectable Tyr-Gly-Gly-Phe-Met but did find significant concentrations of Tyr-Gly-Gly. Using thiorphan, there was no detectable Tyr-Gly-Gly-Phe-Met, but there was a high concentration of Tyr. Which of the following is the best conclusion about Met-enkephalin metabolism that can be drawn from these data?

A. Bestatin inhibits an aminopeptidase, and thiorphan inhibits an endopeptidase in the degradative pathway
B. Bestatin inhibits a carboxypeptidase in the degradative pathway
C. Bestatin inhibits an endopeptidase in the degradative pathway
D. Thiorphan inhibits an aminopeptidase, and bestatin inhibits an endopeptidase in the degradative pathway
E. Thiorphan inhibits an aminopeptidase in the degradative pathway

A

The correct answer is A. Met-enkephalin, the most abundant opioid peptide in the human brain, undergoes two routes of metabolism. One route releases a tripeptide and therefore is the result of a peptidase that cuts an amino acid bond within the molecule: an endopeptidase. The other route releases free tyrosine and therefore is an exopeptidase. Exopeptidases can remove amino acid residues from the amino- or carboxyl-terminus of the protein. By convention, all peptide sequences are given from the N to the C terminus, the direction of translation. Tyrosine is therefore at the amino-terminus of Met-enkephalin, and its release is the result of digestion by an aminopeptidase.
The scientists have used two drugs to highlight the two enzymatic pathways. With bestatin, Met-enkephalin is metabolized only to the tripeptide; therefore bestatin inhibits the aminopeptidase enzyme, preventing release of free tyrosine residues. With thiorphan, Met-enkephalin is metabolized to free tyrosine; the tripeptide is no longer formed. Thiorphan is an inhibitor of the endopeptidase. The lack of persistence of Met-enkephalin in the presence of an enzyme inhibitor is evidence that the peptide's metabolism is shifted in the direction of the noninhibited enzyme. A schematic of the metabolism would be:



Tyrosine cannot be the result of carboxypeptidase activity (choice B), since the carboxyl-terminus of Met-enkephalin is a methionine.

Bestatin inhibits an aminopeptidase, not an endopeptidase (choice C). An endopeptidase would not release a free amino acid residue.

Met-enkephalin is indeed metabolized by an aminopeptidase and an endopeptidase, but bestatin inhibits the aminopeptidase and thiorphan inhibits the endopeptidase (compare with choice D).

Thiorphan does not inhibit an aminopeptidase (choice E); furthermore, such an enzyme would release a free Tyr and a tetrapeptide.